Immunogenic peptides

ABSTRACT

The invention provides relatively short immunogenic peptides, and biologically active variants thereof, associated with leukemia which elicit an immune response. Nucleic acids encoding the immunogenic peptides and antibodies specific for the peptides are also provided. The immunogenic peptides can be included in pharmaceutical compositions, such as cancer vaccines, and used for the treatment of cancer.

FIELD OF INVENTION

[0001] The present invention relates to immunogenic peptides. More particularly the present invention relates to relatively short peptides associated with leukemia that elicit an immune response.

BACKGROUND OF THE INVENTION

[0002] Cancers, including leukemia, are the leading cause of death in humans. Roughly 32,000 new cases of, and 22,000 deaths caused by, leukemia occur in the U.S. each year. Most cases occur of leukemia occur in adults. The exact cause of leukemia is not known, but links between certain activities, such as exposure to carcinogens, and the incidence of certain types of carcinomas, lymphomas, e.g., leukemia and tumors, has been shown by a number of researchers. However, such exposures do not explain most cases of leukemia.

[0003] Many types of chemotherapeutic agents have been shown to be effective against leukemia, but not all types of leukemia cells respond to these agents, and, unfortunately, many of these agents also destroy normal cells. Despite advances in the field of leukemia treatments, the leading therapies to date are radiation, chemotherapy and bone marrow transplants. However, these therapies generally harm normal cells as well as leukemic cells. Ideally cytotoxic agents that have specificity for leukemia cells while only minimally affecting normal healthy cells would be extremely desirable. Unfortunately, none have been found and instead agents which target especially rapidly dividing cells (both diseased and normal) have been used.

[0004] Thus there continues to be a strong need for methods of diagnosing and viable treatment regimens for leukemia.

SUMMARY OF THE INVENTION

[0005] One aspect of the present invention provides isolated leukemic antigens comprising a fragment of CD33 antigen or a variant thereof that is capable of stimulating a cytotoxic T-lymphocyte reaction. The fragment or variant thereof can be 8, 9, 10, 11 or 12 amino acids in length. In some aspects the isolated leukemic antigen is immunologically recognized by MHC restricted T-Lymphocytes that are HLA-A2.1 restricted. The isolated leukemic antigen can have the amino acid sequence YLALCLCLI (SEQ ID NO: 1), AIISGDSPV (SEQ ID NO: 2) or YIISGDSPV (SEQ ID NO: 3) with or without one or more conservative or nonconservative amino acid substitutions. The isolated leukemic antigen can also be combined with one or more co-immunostimulatory molecules.

[0006] The present invention also provides a method for stimulating an immune effector cell response achieved by contacting the isolated leukemic antigen with an immune effector cell which stimulates the immune effector cell to respond against the isolated leukemic antigen. In some methods the immune effector cell is a naive T-lymphocyte or a memory T-lymphocyte. The method can be performed by contacting the isolated leukemic antigen with an antigen presenting cell, in vivo or in vitro, such that the antigen presenting cell contacts the isolated leukemic antigen with the immune effector cell. Suitable antigen presenting cells are dendritic cells or T2 cells.

[0007] The present invention also pertains to immune effector cells and antigen presenting cells produced by these methods. Nucleic acids encoding the present isolated leukemic antigens also form part of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008]FIG. 1A shows IFN-γ production of cytotoxic T-lymphocytes (CTLs) stimulated by dendritic cells pulsed with the AIISGDSPV (SEQ ID NO:2) peptide; and

[0009]FIG. 1B shows IFN-65 production of CTLs stimulated by T2 cells pulsed with the AIISGDSPV (SEQ ID NO:2) peptide.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0010] The present invention encompasses fragments of the CD33 antigen, and variants thereof which are immunologically recognized by T lymphocytes of the immune system. CD33 antigen is generally expressed by myeloblasts, promyelocytes, myeolcytes, mast cells, throughout monocyte differentiation and is also highly expressed on a large percentage of leukemic cells. The present invention further encompasses the antigen cancer epitope(s) which are contained in the tumor antigen. The antigenic cancer epitope specifically causes a cellular mediated immune response by interaction with T cells of the immune system. This interaction between the antigenic cancer epitope and the T cells causes the T cells to respond against, and prevent, eliminate or reduce the cancer in a mammal, including humans. The peptides, nucleic acid molecules which code for such peptides, binding agents such as antibodies, antigen presenting cells (APCs) and/or immune system cells (e.g., T cells), antibodies against such peptides and nucleic acids, are useful, inter alia, in diagnostic and therapeutic contexts. Thus, the present invention provides a cancer vaccine.

[0011] The CD33 antigen is discussed in Simmons et al., J. Immunol. 141 (8), 2797-2800 (1988). Proteins similar or homologous to CD33 are discussed in Tchilian et al. Blood 83 (11), 3188-3198 (1994); Takei et al. Cytogenet. Cell Genet. 78 (3-4), 295-300 (1997). The sequence for the CD33 antigen (SEQ ID NO:4) as reported in Simmons et al. and disclosed in the SwissProt annotated protein record P20138 is as follows:

[0012] 1 mplllllpll wagalamdpn fwlqvqesvt vqeglcvlvp ctffhpipyy dknspvhgyw

[0013] 61 fregaiisgd spvatnkldq evqeetqgrf rllgdpsrnn cslsivdarr rdngsyffrm

[0014] 121 ergstkysyk spqlsvhvtd lthrpkilip gtlepghskn ltcsvswace qgtppifswl

[0015] 181 saaptslgpr tthssvliit prpqdhgtnl tcqvkfagag vttertiqln vtyvpqnptt

[0016] 241 gifpgdgsgk qetragvvhg aiggagvtal lalclcliff ivkthrrkaa rtavgrndth

[0017] 301 pttgsaspkh qkksklhgpt etsscsgaap tvemdeelhy aslnfhgmnp skdtsteyse

[0018] 361 vrtq

[0019] References discussing the CD33 protein, sequence, nucleic acids encoding and antibodies against include: Andrews et al. Blood 62:124-132 (1983); Andrews et al., J Exp Med 169:1721-1731 (1989); Caron et al. Blood 83:1760-1768 (1994); Freeman et al., Blood 85:2005-2012 (1995); Griffin et al., Leuk Res 8:521-534 (1984); Peiper et al., Leukocyte Typing V. Oxford University Press p837-840 (1995); Peiper et al., Blood 72:314-321 (1988); Robertson et al., Blood 79:2229-2236 (1992); Sobol et al., N Engl J Med 316:1111-1117 (1987); and Thomas et al., J Immunol 153:4016-4028 (1994).

Proteins

[0020] The compounds of this invention generally comprise a polypeptide, sometimes in isolated form, that stimulates a Th1 or CTL (cytotoxic T-lymphocyte) immune response in peripheral blood mononuclear cells (PBMCs). In particular, polypeptides 8-12 amino acids in length comprising a stimulatory portion of the CD33 antigen are disclosed, such as a cancer rejection antigen. A cancer rejection antigen is an example of a unique fragment of a cancer specific polypeptide which retains the functional capability of HLA binding and interaction with cytotoxic T lymphocytes. Tumor rejection antigens presented by HLA molecules typically are 9 amino acids in length, although peptides of 8, 9, 10, 11 and 12 and more amino acids can retain the capability to interact with HLA and cytotoxic T lymphocyte to an extent effective to provoke a cytotoxic T lymphocyte response (see, e.g., Van den Eynde & Brichard, Curr. Opin. Immunol. 7:674-681, 1995; Coulie et al., Stem Cells 13:393-403, 1995) and discussed in U.S. Pat. No. 6,271,019. Polypeptides encompass amino acid chains of any length, including full length proteins and portions thereof, wherein amino acid residues are linked by covalent peptide bonds. Although CD33 fragments are described herein for exemplary purposes, portions thereof, and variants of the polypeptide (or portions thereof) can also be used. In one preferred embodiment, the polypeptides are substantially free of contaminating endogenous materials.

[0021] The term “isolated” means separated from constituents, cellular and otherwise, in which the polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, are normally associated with in nature. As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, does not require “isolation” to distinguish it from its naturally occurring counterpart. In addition, a “concentrated,” “separated” or “diluted” polynucleotide, peptide, polypeptide, protein, antibody, or fragment thereof, is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is greater than “concentrated” or less than “separated” than that of its naturally occurring counterpart. A polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, which differs from the naturally occurring counterpart in its primary sequence or for example, by its glycosylation pattern, need not be present in its isolated form since it is distinguishable from its naturally occurring counterpart by its primary sequence, or alternatively, by another characteristic such as glycosylation pattern. Although not explicitly stated for each of the inventions disclosed herein, it is to be understood that all of the above embodiments for each of the compositions disclosed below and under the appropriate conditions, are provided by this invention. Thus, a non-naturally occurring polynucleotide is provided as a separate embodiment from the isolated naturally occurring polynucleotide. A protein produced in a bacterial cell is provided as a separate embodiment from the naturally occurring protein isolated from a eukaryotic cell in which it is produced in nature.

[0022] Several peptide fragments of the CD33 antigen have been discovered which have the ability to stimulate a T-lymphocyte mediated cellular immune response. Peptides of the present invention can include these amino acid sequences in any configuration or location in the peptide.

[0023] Biologically functionally equivalent variants of the present CD33 polypeptide fragments, i.e., variants of polypeptides which retain the function of the natural polypeptide fragment, can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. The skilled artisan will also realize that conservative amino acid substitutions can be made in the present polypeptides to provide such functionally active homologs of the forgoing polypeptides, i.e., the homologs retain the functional capabilities of the polypeptides. As used herein, a “conservative amino acid substitution” refers to an amino acid substitution which does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Conservative substitutions of amino acids generally are understood to include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. The present invention also encompasses polypeptides with one or more nonconservative amino acid substitutions that retain similar functionality compared to the non-modified peptide. Generally a “nonconservative amino acid substitution” is understood to be an amino acid substituted by an alternative amino acid of differing charge density, hydrophilicity/hydrophobicity, size, and/or configuration (e.g., Val for Phe). The means of making such modifications are well known in the art and can be readily accomplished by means of commercially available kits and vectors (e.g., New England Biolabs, Inc., Beverly, Mass.; Clontech, Palo Alto, Calif.). Moreover, the means of assessing such substitutions (e.g., in terms of effect on ability to bind and enter cells) are known in the art and described for example in U.S. Pat. No. 6,329,190.

[0024] The polypeptides of the present invention also include variants of the CD33 fragments that retain the ability to stimulate a Th1 or CTL immune response in PBMCs. Such variants include various structural forms of the primary protein, including related and homologous proteins which can be found in non-human species. Due to the presence of ionizable amino and carboxyl groups, for example, a polypeptide fragment can be in the form of an acidic or basic salt, or can be in neutral form. Individual amino acid residues can also be modified by oxidation or reduction.

[0025] Variants within the scope of this invention also include polypeptides in which the primary amino acid structure of the polypeptide fragment is modified by forming covalent or aggregative conjugates with other polypeptides or chemical moieties such as glycosyl groups, lipids, phosphate, acetyl groups and the like. Covalent derivatives can be prepared, for example, by linking particular functional groups to amino acid side chains or at the N- or C-termini. Alternatively, for derivatives in which a polypeptide is joined to a CD33 fragment, a fusion protein can be prepared using recombinant DNA techniques, as described below. As will be understood by the skilled artisan, fusion peptides containing the 8-12 amino acid CD33 fragment of the present invention are not limited to 8-12 amino acids in total size, but instead that the immunogenic fragment of the CD33 antigen is 8-12 residues in size. In one such embodiment, the CD33 polypeptide polypeptide can be conjugated to a signal (or leader) polypeptide sequence at the N-terminal region of the protein which co-translationally or post-translationally directs transfer of the protein from its site of synthesis to its site of function inside or outside of the cell membrane or wall (e.g., the yeast α-factor leader).

[0026] Also provided by this application are the polypeptides and proteins described herein conjugated to a detectable agent for use in the diagnostic methods. For example, detectably labeled proteins and polypeptides can be bound to a column and used for the detection and purification of antibodies. They also are useful as immunogens for the production of antibodies as described below. The proteins and fragments of this invention are useful in an in vitro assay system to screen for agents or drugs, which modulate cellular processes.

[0027] Such detectable agents include protein fusions with other proteins and which can facilitate purification or identification of the polypeptides (e.g., poly-His). For example, the peptide described by Hopp et al., Bio/Technology 6:1204 (1988) is a highly antigenic peptide that can be used to facilitate identification. Such a peptide provides an epitope reversibly bound by a specific monoclonal antibody, enabling rapid assay and facile purification of expressed recombinant protein. The sequence of Hopp et al. is also specifically cleaved by bovine mucosal enterokinase, allowing removal of the peptide from the purified protein. Fusion proteins capped with such peptides can also be resistant to intracellular degradation in E. coli.

[0028] Protein fusions encompassed by this invention further include, for example, the polypeptides linked to an immunoglobulin Fe region. If the fusion proteins are made with both heavy and light chains of an antibody, it is possible to form a protein oligomer with as many as four CD33 protein fragment regions. Also within the scope of the present invention are polypeptides linked to a leucine zipper domain. Leucine zipper domains are described, for example, in published PCT Application WO 94/10308. The present polypeptides comprising leucine zippers may, for example, be oligomeric, dimeric or trimeric. All of the above protein fusions can be prepared by chemical linkage or as fusion proteins, as described in U.S. Pat. No. 6,013,268. Preferred protein fusions include polypeptides that comprise sequences useful for stimulating immunity to infectious pathogens (e.g., antigens). Such sequences can be derived, for example, from viruses, tumor cells, parasites or bacteria.

[0029] The proteins and polypeptides of this invention can be obtained by chemical synthesis using a commercially available automated peptide synthesizer such as those manufactured by Perkin Elmer/Applied Biosystems, Inc., Model 430A or 431A, Foster City, Calif., USA. The synthesized protein or polypeptide can be precipitated and further purified, for example by high performance liquid chromatography (HPLC). Accordingly, this invention also provides a process for chemically synthesizing the proteins of this invention by providing the sequence of the protein and reagents, such as amino acids and enzymes and linking together the amino acids in the proper orientation and linear sequence.

[0030] Alternatively, the proteins and polypeptides can be obtained by well-known recombinant methods as described above using the host cell and vector systems described above.

[0031] Nonpeptide analogs of peptides, e.g., those which provide a stabilized structure or lessened biodegradation, are also contemplated. Peptide mimetic analogs can be prepared based on a selected binding peptide by replacement of one or more residues by nonpeptide moieties. Preferably, the nonpeptide moieties permit the peptide to retain its natural conformation, or stabilize a preferred, e.g., bioactive, confirmation. Such peptides can be tested in molecular or cell-based binding assays to assess the effect of the substitution(s) on conformation and/or activity. One example of methods for preparation of nonpeptide mimetic analogs from peptides is described in Nachman et al., Regul. Pept. 57:359-370 (1995) and disclosed in U.S. Pat. No. 6,291,430.

[0032] The proteins of this invention also can be combined with various liquid phase carriers, such as sterile or aqueous solutions, pharmaceutically acceptable carriers, suspensions and emulsions. Examples of non-aqueous solvents include propyl ethylene glycol, polyethylene glycol and vegetable oils. When used to prepare antibodies, the carriers also can include an adjuvant that is useful to non-specifically augment a specific immune response. A skilled artisan can easily determine whether an adjuvant is required and select one. However, for the purpose of illustration only, suitable adjuvants include, but are not limited to Freund's Complete and Incomplete, mineral salts and polynucleotides.

Nucleic Acids

[0033] Polynucleotides of the subject invention generally comprise a DNA or RNA sequence that encodes all or a portion of the above CD33 polypeptide fragments, or that is complementary to such a sequence. Nucleic acids encoding proteins related or homologous to CD33 are disclosed in Tchilian et al. Blood 83 (11), 3188-3198 (1994); and Takei et al. Cytogenet. Cell Genet. 78 (3-4), 295-300 (1997). Preferably, the CD33 polypeptide fragment is a leukemia associated nucleic acid or polypeptide is a nucleic acid or polypeptide expressed preferentially in leukemias and solid forms of leukemia cell malignancies, such as lymphomas. Various methods for determining the expression of a nucleic acid and/or a polypeptide in normal and leukemia cells are known to those of skill in the art.

[0034] The reported nucleic acid sequence encoding the CD33 protein described above, as reported in Simmons et al., J. Immunol. 141 (8), 2797-2800 (1988), is (SEQ ID NO: 5): 1 gcttcctcag acatgccgct gctgctactg ctgcccctgc tgtgggcagg ggccctggct 61 atggatccaa atttctggct gcaagtgcag gagtcagtga cggtacagga gggtttgtgc 121 gtcctcgtgc cctgcacttt cttccatccc ataccctact acgacaagaa ctccccagtt 181 catggttact ggttccggga aggagccatt atatccgggg actctccagt ggccacaaac 241 aagctagatc aagaagtaca ggaggagact cagggcagat tccgcctcct tggggatccc 301 agtaggaaca actgctccct gagcatcgta gacgccagga ggagggataa tggttcatac 361 ttctttcgga tggagagagg aagtaccaaa tacagttaca aatctcccca gctctctgtg 421 catgtgacag acttgaccca caggcccaaa atcctcatcc ctggcactct agaacccggc 481 cactccaaaa accttacctg ctctgtgtcc tgggcctgtg agcagggaac acccccgatc 541 ttctcctggt tgtcagctgc ccccacctcc ctgggcccca ggactactca ctcctcggtg 601 ctcataatca ccccacggcc ccaggaccac ggcaccaacc tgacctgtca ggtgaagttc 661 gctggagctg gtgtgactac ggagagaacc atccagctca acgtcaccta tgttccacag 721 aacccaacaa ctggtatctt tccaggagat ggctcaggga aacaagagac cagagcagga 781 ctggttcatg gggccattgg aggagctggt gttacagccc tgctcgctct ttgtctctgc 841 ctcatcttct tcatagtgaa gacccacagg aggaaagcag ccaggacagc agtgggcagc 901 aatgacaccc accctaccac agggtcagcc tccccgaaac accagaagaa ctccaagtta 961 catggcccca ctgaaacctc aagctgttca ggtgccgccc ctactgtgga gatggatgag 1021 gagctgcatt atgcttccct caactttcat gggatgaatc cttccaagga cacctccacc 1081 gaatactcag aggtcaggac ccagtgagga accctcaaga gcatcaggct cagctagaag 1141 atccacatcc tctacaggtc ggggaccaaa ggctgattct tggagattta actccccaca 1201 ggcaatgggt ttatagacat tatgtgagtt tcctgctata ttaacatcat cttgagactt 1261 tgcaagcaga gagtcgtgga atcaaatctg tgctctttca tttgctaagt gtatgatgtc 1321 acacaagctc cttaaccttc catgtctcca ttttcttctc tgtgaagtag gtataagaag 1381 tcctatctca tagggatgct gtgagcatta aataaaggta cacatggaaa acaccag

[0035] The nucleic acids contemplate the degeneracy of the genetic code in which nucleic acids can be coded by alternative codons to those present in the native materials. For example, serine residues are encoded by the codons TCA, AGT, TCC, TCG, TCT and AGC. Each of the six codons is equivalent for the purposes of encoding a serine residue. Thus, it will be apparent to one of ordinary skill in the art that any of the serine-encoding nucleotide triplets can be employed to direct the protein synthesis apparatus, in vitro or in vivo, to incorporate a serine residue. Similarly, nucleotide sequence triplets which encode other amino acid residues include, but are not limited to: CCA, CCC, CCG and CCT (proline codons); CGA, CGC, CGG, CGT, AGA and AGG (arginine codons); ACA, ACC, ACG and ACT (threonine codons); AAC and AAT (asparagine codons); and ATA, ATC and ATT (isoleucine codons). Other amino acid residues can be encoded similarly by multiple nucleotide sequences. Thus, the invention embraces degenerate nucleic acids that differ from the biologically isolated nucleic acids in codon sequence due to the degeneracy of the genetic code.

[0036] Further contemplated are antisense oligonucleotides that selectively bind to a leukemia associated gene nucleic acid molecule. Additionally, nucleic acid mimetics, such as peptide nucleic acids are contemplated in the definition of nucleic acids.

[0037] The polynucleotides and peptides can be used for comparison to known and unknowns sequences using a computer-based method to match a sample sequence with known sequences. Thus, this invention also provides the polynucleotides or peptides in a computer database or in computer readable form, including applications utilizing the internet.

[0038] A linear search through such a database can be used. Alternatively, the polynucleotide sequence can be converted into a unique numeric representation. The comparison aspects can be implemented in hardware or software, or a combination of both. Preferably, these aspects of the invention are implemented in computer programs executing on a programmable computer comprising a processor, a data storage system (including volatile and non-volatile memory and/or storage elements), at least one input device, and at least one output device. Data input through one or more input devices for temporary or permanent storage in the data storage system includes sequences, and can include previously generated polynucleotides and codes for known and/or unknown sequences. Program code is applied to the input data to perform the functions described above and generate output information. The output information is applied to one or more output devices, in known fashion.

[0039] Each such computer program is preferably stored on a storage media or device (e.g., ROM or magnetic diskette) readable by a general or special purpose programmable computer, for configuring and operating the computer when the storage media or device is read by the computer to perform the procedures described herein. The inventive system can also be considered to be implemented as a computer-readable storage medium, configured with a computer program, where the storage medium so configured causes a computer to operate in a specific and predefined manner to perform the functions described herein.

[0040] The polynucleotides of the present invention also can serve as primers for the detection of genes or gene transcripts that are expressed in APC, for example, to confirm transduction of the polynucleotides into host cells. In this context, amplification means any method employing a primer-dependent polymerase capable of replicating a target sequence with reasonable fidelity. Amplification can be carried out by natural or recombinant DNA-polymerases such as T7 DNA polymerase, Klenow fragment of E. coli DNA polymerase, and reverse transcriptase.

[0041] The invention further provides the isolated polynucleotide operatively linked to a promoter of RNA transcription, as well as other regulatory sequences for replication and/or transient or stable expression of the DNA or RNA. As used herein, the term “operatively linked” means positioned in such a manner that the promoter will direct transcription of RNA off the DNA molecule. Examples of such promoters are SP6, T4 and T7. In certain embodiments, cell-specific promoters are used for cell-specific expression of the inserted polynucleotide. Vectors which contain a promoter or a promoter/enhancer, with termination codons and selectable marker sequences, as well as a cloning site into which an inserted piece of DNA can be operatively linked to that promoter are well known in the art and commercially available. For general methodology and cloning strategies, see GENE EXPRESSION TECHNOLOGY (Goeddel ed., Academic Press, Inc. (1991)) and references cited therein and VECTORS: ESSENTIAL DATA SERIES (Gacesa and Ramji, eds., John Wiley & Sons, N.Y. (1994)), which contains maps, functional properties, commercial suppliers and a reference to GenEMBL accession numbers for various suitable vectors. Preferable, these vectors are capable of transcribing RNA in vitro or in vivo.

[0042] Expression vectors containing these nucleic acids are useful to obtain host vector systems to produce proteins and polypeptides. It is implied that these expression vectors must be replicable in the host organisms either as episomes or as an integral part of the chromosomal DNA. Suitable expression vectors include plasmids, viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, cosmids, etc. Adenoviral vectors are particularly useful for introducing genes into tissues in vivo because of their high levels of expression and efficient transformation of cells both in vitro and in vivo. When a nucleic acid is inserted into a suitable host cell, e.g., a prokaryotic or a eukaryotic cell and the host cell replicates, the protein can be recombinantly produced. Suitable host cells will depend on the vector and can include mammalian cells, animal cells, human cells, simian cells, insect cells, yeast cells, and bacterial cells constructed using well known methods. See Sambrook, et al. (1989) Supra. In addition to the use of viral vector for insertion of exogenous nucleic acid into cells, the nucleic acid can be inserted into the host cell by methods well known in the art such as transformation for bacterial cells; transfection using calcium phosphate precipitation for mammalian cells; or DEAE-dextran; electroporation; or microinjection. See Sambrook et al. (1989) Supra for this methodology. Thus, this invention also provides a host cell, e.g. a mammalian cell, an animal cell (rat or mouse), a human cell, or a prokaryotic cell such as a bacterial cell, containing a polynucleotide encoding a protein or polypeptide or antibody.

[0043] When the vectors are used for gene therapy in vivo or ex vivo, a pharmaceutically acceptable vector is preferred, such as a replication-incompetent retroviral or adenoviral vector. Pharmaceutically acceptable vectors containing the nucleic acids of this invention can be further modified for transient or stable expression of the inserted polynucleotide. As used herein, the term “pharmaceutically acceptable vector” includes, but is not limited to, a vector or delivery vehicle having the ability to selectively target and introduce the nucleic acid into dividing cells. An example of such a vector is a “replication-incompetent” vector defined by its inability to produce viral proteins, precluding spread of the vector in the infected host cell. An example of a replication-incompetent retroviral vector is LNL6 (Miller, A. D. et al. (1989) BioTechniques 7:980-990). The methodology of using replication-incompetent retroviruses for retroviral-mediated gene transfer of gene markers is well established (Correll, et al. (1989) PNAS USA 86:8912; Bordignon (1989) PNAS USA 86:8912-52; Culver, K. (1991) PNAS USA 88:3155; and Rill, D. R. (1991) Blood 79(10):2694-700.

[0044] These host cells containing the polynucleotides of this invention are useful for the recombinant replication of the polynucleotides and for the recombinant production of peptides. Alternatively, the cells can be used to induce an immune response in a subject in the methods described herein. When the host cells are antigen presenting cells, they can be used to expand a population of immune effector cells such as tumor infiltrating lymphocytes which in turn are useful in adoptive immunotherapies.

Protein Binding Agents (Antibodies)

[0045] The invention also involves agents which bind to leukemia associated polypeptides disclosed herein. Such binding partners can be used in screening assays to detect the presence or absence of the present polypeptides and in purification protocols to isolate these polypeptides. Likewise, such binding partners can be used to selectively target drugs, toxins or other molecules to leukemia cells which present the associated polypeptides. In this manner, cells present in solid or non-solid tumors which express the CD33 fragments can be treated with cytotoxic compounds.

[0046] The invention, therefore, involves antibodies or fragments of antibodies having the ability to selectively bind to the disclosed polypeptides. Antibodies against the CD33 antigen are discussed in Co et al., Chimeric and Humanized Antibodies with Specificity for the CD33 Antigen, J. Immunol. 148, 1149-1154 (1992). Antibodies include polyclonal and monoclonal antibodies, prepared according to conventional methodology. The antibodies can include, but are not limited to mouse, rat, and rabbit or human antibodies. The antibodies are useful to identify and purify polypeptides and APCs expressing the polypeptides.

[0047] The antibodies of the present invention are prepared by any of a variety of methods, including administering protein, fragments of protein, cells expressing the protein or fragments thereof and the like to an animal to induce polyclonal antibodies. Laboratory methods for producing polyclonal antibodies and monoclonal antibodies, as well as deducing their corresponding nucleic acid sequences, are known in the art, see Harlow and Lane (1988) Supra and Sambrook, et al. (1989) Supra. The monoclonal antibodies of this invention can be biologically produced by introducing protein or a fragment thereof into an animal, e.g., a mouse or a rabbit. The antibody producing cells in the animal are isolated and fused with myeloma cells or heteromyeloma cells to produce hybrid cells or hybridomas. Accordingly, the hybridoma cells producing the monoclonal antibodies of this invention also are provided.

[0048] The antibodies of this invention can be linked to a detectable agent or label. There are many different labels and methods of labeling known to those of ordinary skill in the art. The coupling of antibodies to low molecular weight haptens can increase the sensitivity of the assay. The haptens can then be specifically detected by means of a second reaction. For example, it is common to use haptens such as biotin, which reacts avidin, or dinitropherryl, pyridoxal, and fluorescein, which can react with specific anti-hapten antibodies. See Harlow and Lane (1988) Supra. Antibodies also can be coupled to specific labeling agents for imaging or to antitumor agents, including, but not limited to, methotrexate, radioiodinated compounds, toxins such as ricin, other cytostatic or cytolytic drugs, and so forth. Antibodies prepared according to the invention also preferably are specific for the CD33 complexes described herein. Variations of antibodies encompasses by the present invention can be found in U.S. Pat. No. 6,303,756.

[0049] The monoclonal antibodies of the invention also can be bound to many different carriers. Thus, this invention also provides compositions containing the antibodies and another substance, active or inert. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magnetite. The nature of the carrier can be either soluble or insoluble for purposes of the invention. Those skilled in the art will know of other suitable carriers for binding monoclonal antibodies, or will be able to ascertain such, using routine experimentation.

[0050] Thus, using the protein or fragment thereof, and well known methods, one of skill in the art can produce and screen the hybridoma cells and antibodies of this invention for antibodies having the ability to bind the proteins or polypeptides. As detailed herein, such antibodies can also be used to identify tissues expressing protein or to purify protein.

[0051] If a monoclonal antibody being tested binds with the protein or polypeptide, then the antibody being tested and the antibodies provided by the hybridomas of this invention are equivalent. It also is possible to determine without undue experimentation, whether an antibody has the same specificity as the monoclonal antibody of this invention by determining whether the antibody being tested prevents a monoclonal antibody of this invention from binding the protein or polypeptide with which the monoclonal antibody is normally reactive. If the antibody being tested competes with the monoclonal antibody of the invention as shown by a decrease in binding by the monoclonal antibody of this invention, then it is likely that the two antibodies bind to the same or a closely related epitope. Alternatively, one can pre-incubate the monoclonal antibody of this invention with a protein with which it is normally reactive, and determine if the monoclonal antibody being tested is inhibited in its ability to bind the antigen. If the monoclonal antibody being tested is inhibited then, in all likelihood, it has the same, or a closely related, epitopic specificity as the monoclonal antibody of this invention.

[0052] The term “antibody” also is intended to include antibodies of all isotypes. Particular isotypes of a monoclonal antibody can be prepared either directly by selecting from the initial fusion, or prepared secondarily, from a parental hybridoma secreting a monoclonal antibody of different isotype by using the sib selection technique to isolate class switch variants using the procedure described in Steplewski, et al. (1985) Proc. Natl. Acad. Sci. 82:8653 or Spira, et al. (1984) J. Immunol. Methods 74:307.

[0053] This invention also provides biological active fragments of the polyclonal and monoclonal antibodies described above. These “antibody fragments” retain some ability to selectively bind with its antigen or immunogen. Such antibody fragments can include, but are not limited to: (1) Fab, (2) Fab′, (3) F(ab′)₂, (4) Fv, and (5) SCA. A specific example of “a biologically active antibody fragment” is a CDR region of the antibody. Methods of making these fragments are known in the art, see for example, Harlow and Lane (1988) Supra.

[0054] The isolation of other hybridomas secreting monoclonal antibodies with the specificity of the monoclonal antibodies of the invention can also be accomplished by one of ordinary skill in the art by producing anti-idiotypic antibodies (Herlyn, et al. (1986) Science 232:100). An anti-idiotypic antibody is an antibody which recognizes unique determinants present on the monoclonal antibody produced by the hybridoma of interest.

[0055] Idiotypic identity between monoclonal antibodies of two hybridomas demonstrates that the two monoclonal antibodies are the same with respect to their recognition of the same epitopic determinant. Thus, by using antibodies to the epitopic determinants on a monoclonal antibody it is possible to identify other hybridomas expressing monoclonal antibodies of the same epitopic specificity.

[0056] Compositions containing the antibodies, fragments thereof or cell lines which produce the antibodies, are encompassed by this invention. When these compositions are to be used pharmaceutically, they can be combined with a pharmaceutically acceptable carrier.

Pulsing Antigen Presenting Cells

[0057] The polypeptides of this invention also can be pulsed into antigen presenting cells using the methods described herein either in vivo or in vitro. Various methods of pulsing the antigen presenting cells are disclosed in U.S. Pat. No. 6,306,640, Lodge et al. (2000), Cancer Res. 60:829, Lau et al. (2001), J Immun. 24(1):66, Gajewski et al. (2001), Clin. Cancer Res. 7:895s, Morse et al. (1999), Clin. Cancer Res. 5:1331 and Schmidt et al. (1997) Proc. Natl. Acad. Sci. 94:3262. Antigen-presenting cells, include, but are not limited to dendritic cells (DCs), monocytes/macrophages, B lymphocytes or other cell type(s) expressing the necessary MHC/co-stimulatory molecules. The methods described below focus primarily on DCs which are the most potent, preferred APCs. These host cells containing the polypeptides or proteins are further provided.

[0058] The terms “antigen-presenting cells” or “APCs” includes both intact, whole cells as well as other molecules which are capable of inducing the presentation of one or more antigens, preferably in association with MHC molecules. Examples of suitable APCs are discussed in detail below and include, but are not limited to, whole cells such as macrophages, dendritic cells, B cells, purified MHC class I molecules complexed to beta 2-microglobulin; and foster antigen presenting cells.

[0059] Dendritic cells (DCs) are potent antigen-presenting cells. It has been shown that DCs provide all the signals required for T cell activation and proliferation. These signals can be categorized into two types. The first type, which gives specificity to the immune response, is mediated through interaction between the T-cell receptor/CD3 (“TCR/CD3”) complex and an antigenic peptide presented by a major histocompatibility complex (“MHC”) class I or II protein on the surface of APCs. This interaction is necessary, but not sufficient, for T cell activation to occur. In fact, without the second type of signals, the first type of signals can result in T cell anergy. The second type of signals, called co-stimulatory signals, is neither antigen-specific nor MHC-restricted, and can lead to a full proliferation response of T cells and induction of T cell effector functions in the presence of the first type of signals. As used herein, “dendritic cell” is to include, but not be limited to a pulsed dendritic cell, a foster cell or a dendritic cell hybrid.

[0060] The term “immune effector cells” refers to cells capable of binding an antigen or which mediate an immune response. These cells include, but are not limited to, T cells, B cells, monocytes, macrophages, NK cells and cytotoxic T lymphocytes (CTLs), for example CTL lines, CTL clones, and CTLs from tumor, inflammatory, or other infiltrates. A “naive” cell is a cell that has never been exposed to an antigen.

[0061] Isolated host cells which present the polypeptides of this invention in the context of MHC molecules are further useful to expand and isolate a population of educated, antigen-specific immune effector cells. The immune effector cells, e.g., cytotoxic T lymphocytes, are preferably produced by culturing naive immune effector cells with antigen-presenting cells that present the polypeptides in the context of MHC molecules on the surface of the APCs. The population can be purified using methods known in the art, e.g., FACS analysis or ficoll gradient. The methods to generate and culture the immune effector cells as well as the populations produced thereby also are the inventor's contribution and invention. Pharmaceutical compositions comprising the cells and pharmaceutically acceptable carriers are useful in adoptive immunotherapy. Prior to administration in vivo, the immune effector cells can be screened in vitro for their ability to lyse melanoma tumor cells.

[0062] In one embodiment, the immune effector cells and/or the APCs are genetically modified. Using standard gene transfer, genes coding for co-stimulatory molecules and/or stimulatory cytokines can be inserted prior to, concurrent to or subsequent to expansion of the immune effector cells.

Immune Effector Cells

[0063] The present invention also encompasses these immune effector cells that have been exposed to polypeptides of the present invention, preferably in an isolated form. Alternative to the above, the immune effector cells can be exposed to the polypeptides, preferably in the presence of one or more stimulatory molecules, without the help of antigen presenting cells.

Immune Response Induction

[0064] This invention also provides methods of inducing an immune response in a subject, comprising administering to the subject an effective amount of the polypeptides described above under the conditions that induce an immune response to the polypeptide. The polypeptides can be administered in formulations or as polynucleotides encoding the polypeptides. The polynucleotides can be administered in a gene delivery vehicle or by inserting into a host cell which in turn recombinantly transcribes, translates and processes the encoded polypeptide. Isolated host cells containing the polynucleotides of this invention in a pharmaceutically acceptable carrier can therefore combined with appropriate and effective amount of an adjuvant, cytokine or co-stimulatory molecule for an effective vaccine regimen. The vaccination can either be prophylactic or for treatment of established cancer. In one embodiment, the host cell is an APC such as a dendritic cell. The host cell can be further modified by inserting a polynucleotide coding for an effective amount of either or both of a cytokine a co-stimulatory molecule.

[0065] The methods of this invention can be further modified by co-administering an effective amount of a cytokine or co-stimulatory molecule. As used herein, the term “cytokine” refers to any one of the numerous factors that exert a variety of effects on cells, for example, inducing growth or proliferation. Non-limiting examples of cytokines which can be used alone or in combination in the practice of the present invention include, interleukin-2 (IL-2), stem cell factor (SCF), interleukin 3 (IL-3), interleukin 6 (IL-6), interleukin 12 (IL-12), G-CSF, granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin-1 alpha (IL-1.sub.I), interleukin-11 (IL-11), MIP-1_(I), leukemia inhibitory factor (LIF), c-kit ligand, thrombopoietin (TPO) and flt3 ligand. The present invention also includes culture conditions in which one or more cytokine is specifically excluded from the medium. Cytokines are commercially available from several vendors such as, for example, Genzyme (Framingham, Mass.), Genentech (South San Francisco, Calif.), Amgen (Thousand Oaks, Calif.), R&D Systems and Immunex (Seattle, Wash.). It is intended, although not always explicitly stated, that molecules having similar biological activity as wild-type or purified cytokines (e.g., recombinantly produced or muteins thereof) are intended to be used within the spirit and scope of the invention.

[0066] “Co-stimulatory molecules” are involved in the interaction between receptor-ligand pairs expressed on the surface of antigen presenting cells and T cells. One exemplary receptor-ligand pair is the B7 co-stimulatory molecules on the surface of DCs and its counter-receptor CD28 or CTLA-4 on T cells (Freeman, et al. (1993) Science 262:909-911; Young, et al. (1992) J. Clin. Invest. 90: 229; Nabavi, et al. Nature 360:266). Other important co-stimulatory molecules are CD40, CD54, CD80, CD86.

[0067] Patient T cell assays can generally be performed by treating patient PBMCs with the reactive antigens and analyzing the cells for a suitable response. For example, the PBMC supernatant can be assayed for the level of secreted cytokines. Preferably, the cytokine assayed is interferon-gamma, interleukin-2, interleukin-12 (either the p40 subunit or biologically active p70), interleukin-1 or tumor necrosis factor-cc. The cytokines interleukin-4 and interleukin-10 can also be assayed, since the levels of these representative Th2-type cytokines generally decrease in response to treatment with a polypeptide as described herein. Cytokines can be assayed, for example, using commercially available antibodies specific for the cytokine of interest in an ELISA format, with positive results determined according to the manufacturer's instructions. Suitable antibodies can be obtained, for example, from Chemicon, Temucula, Calif. and PharMingen, San Diego, Calif. Alternatively, the treated PBMCs can be assayed for mRNA encoding one or more of the cytokines interferon-gamma, interleukin-2, interleukin-12 p40 subunit, interleukin-1 or tumor necrosis factor-α, or the PBMCs can be assayed for a proliferative response as described herein. Alternatively, cytokines can be measured by testing PBMC supernatants for cytokine-specific biological activities.

Method of Diagnosis

[0068] According to one aspect of the invention, methods for diagnosing a disorder that is characterized by expression of a leukemia associated nucleic acid or polypeptide are provided. The methods involve contacting a biological sample isolated from a subject with an agent specific for the leukemia associated nucleic acid or polypeptide to detect the presence of the leukemia associated nucleic acid or polypeptide in the biological sample. As used herein, “contacting” means placing the biological sample in sufficient proximity to the agent and under the appropriate conditions of, e.g., concentration, temperature, time, ionic strength, to allow the specific interaction between the agent and leukemia associated nucleic acid or polypeptide that are present in the biological sample. In general, the conditions for contacting the agent with the biological sample are conditions known by those of ordinary skill in the art to facilitate a specific interaction between a molecule and its cognate (e.g., a protein and its receptor cognate, an antibody and its protein antigen cognate, a nucleic acid and its complementary sequence cognate) in a biological sample. Exemplary conditions for facilitating a specific interaction between a molecule and its cognate are described in U.S. Pat. No. 5,108,921, issued to Low et al.

[0069] The biological sample can be located in vivo or in vitro. For example, the biological sample can be a hematopoietic tissue in vivo and the agent specific for the leukemia associated nucleic acid or polypeptide can be used to detect the presence of such molecules in the hematopoietic tissue (e.g., for imaging portions of the hematopoietic tissue that express the leukemia associated gene products). Alternatively, the biological sample can be located in vitro (e.g., a blood sample, bone marrow biopsy, tissue extract). In a particularly preferred embodiment, the biological sample can be a cell-containing sample, more preferably a sample containing hematopoietic cells.

[0070] The skilled artisan can determine which HLA molecule binds to the CD33 fragments by, e.g., experiments utilizing antibodies to block specifically individual HLA class I molecules. For example, antibodies which bind selectively to HLA-A2 will prevent efficient presentation of antigens specifically presented by HLA-A2. Thus, if the present peptides are presented by HLA-A2, then the inclusion of anti-HLA-A2 antibodies in an in vitro assay will block the presentation of these antigens. An assay for determining the nature of the HLA molecule is found in U.S. Pat. No. 5,939,526.

Vaccine

[0071] The present invention also provides vaccine compositions comprising the CD33 antigen peptide fragments or nucleic acids encoding these fragments described above. Vaccines can also be prepared from antigen presenting cells that have been pulsed with the peptides or nucleic acids or immune effector cells which have been exposed to the peptides or nucleic acids. The vaccine can contain a single peptide or a range of peptides which cover different or similar epitopes. In addition or alternatively, the vaccine can be a polyvalent vaccine where a single polypeptide can be provided with multiple epitopes.

[0072] In one embodiment the peptide is conjugated to a carrier protein, such as for example a polycation (poly-L-Lysine or poly-L-arginine), tetanus toxoid, diphtheria toxoid or oxidised KLH in order to stimulate T cell help as disclosed in U.S. Pat. No. 6,344,203.

[0073] Included as part of the vaccine, substances which potentiate the immune response can be administered with nucleic acid or peptide components in a cancer vaccine. Such immune response potentiating compounds can be classified as either adjuvants or cytokines. Adjuvants can enhance the immunological response by providing a reservoir of antigen (extracellularly or within macrophages), activating macrophages and stimulating specific sets of lymphocytes. Adjuvants of many kinds are well known in the art; specific examples include MPL (SmithKline Beecham), a congener obtained after purification and acid hydrolysis of Salmonella Minnesota Re 595 lipopolysaccharide, QS21 (SmithKline Beecham), a pure QA-21 saponin purified from Quillja saponaria extract, and various water-in-oil emulsions prepared from biodegradable oils such as squalene and/or tocopherol. Cytokines are also useful in vaccination protocols as a result of lymphocyte stimulatory properties. Many cytokines useful for such purposes will be known to one of ordinary skill in the art, including interleukin-12 (IL-12) which has been shown to enhance the protective effects of vaccines (Science 268: 1432-1434, 1995).

[0074] When administered, the therapeutic compositions of the present invention are administered in pharmaceutically acceptable preparations. Such preparations can routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents.

[0075] The term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. The term “physiologically acceptable” refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism. The characteristics of the carrier will depend on the route of administration. Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials which are well known in the art.

[0076] The therapeutics of the invention can be administered by any conventional route, including injection or by gradual infusion over time. Initial doses can also be followed by booster doses, following immunization protocols standard in the art. The administration may, for example, be oral, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, or transdermal. When antibodies are used therapeutically, a preferred route of administration is by pulmonary aerosol. Techniques for preparing aerosol delivery systems containing antibodies are well known to those of skill in the art. Generally, such systems should utilize components which will not significantly impair the biological properties of the antibodies, such as the paratope binding capacity (see, for example, Sciarra and Cutie, “Aerosols,” in Remington's Pharmaceutical Sciences, 18th edition, 1990, pp 1694-1712). Those of skill in the art can readily determine the various parameters and conditions for producing antibody aerosols without resort to undue experimentation. When using antisense preparations of the invention, slow intravenous administration is preferred.

[0077] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives can also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

[0078] In all cases where cells are used as a vaccine, these can be cells transfected with coding sequences for one or both of the components necessary to provoke a CTL response, or be cells which already express both molecules without the need for transfection. Vaccines also encompass naked DNA or RNA, encoding the present peptides, which can be produced in vitro and administered via injection, particle bombardment, nasal aspiration and other methods. Vaccines of the “naked nucleic acid” type have been demonstrated to provoke an immunological response including generation of CTLs specific for the peptide encoded by the naked nucleic acid (Science 259:1745-1748, 1993). When “disorder” is used herein, it refers to any pathological condition where the tumor rejection antigen precursor is expressed. An example of such a disorder is cancer, leukemias and lymphomas in particular.

[0079] The peptides of the present invention can also be used to elicit or enhance an immune response to an antigen encoded by a DNA vaccine. DNA vaccines encode one or more immunostinulating antigens, such that the antigen is generated in situ. For instance, the DNA vaccine can encode a tumor antigen and, optionally, a peptide as described herein. In such vaccines, the DNA can be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacteria and viral expression systems. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter). Bacterial delivery systems involve the administration of a bacterium (such as Bacillus-Calmette-Guerrin) that expresses an epitope of a leukemia cell antigen on its cell surface. The DNA can be introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus), which can involve the use of a non-pathogenic (defective), replication competent virus. Suitable systems are disclosed, for example, in Fisher-Hoch et al., PNAS 86:317-321, 1989; Flexner et al., Ann. N.Y. Acad. Sci. 569:86-103, 1989; Flexner et al., Vaccine 8:17-21, 1990; U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner, Biotechniques 6:616-627, 1988; Rosenfeld et al., Science 252:431-434, 1991; Kolls et al., PNAS 91:215-219, 1994; Kass-Eisler et al., PNAS 90:11498-11502, 1993; Guzman et al., Circulation 88:2838-2848, 1993; and Guzman et al., Cir. Res. 73:1202-1207, 1993. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA can also be “naked,” as described, for example, in published PCT application WO 90/11092, and Ulmer et al., Science 259:1745-1749, 1993, reviewed by Cohen, Science 259:1691-1692, 1993. The uptake of naked DNA can be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells.

Method of Treatment

[0080] The present invention provides a method of treating individuals suffering from leukemia. In such methods, the introduction of peptides, nucleic acids, protein binding agents, antigen presenting cells and/or immune effector cells as described above serves as an immunotherapeutic, directing and promoting the immune system of the individual to combat leukemic cells that display the CD33 antigen fragments. The methods can comprise administering an effective amount of any of the above compounds to a patient in need of such treatment through the means described above.

[0081] Individuals at risk of developing leukemia, such as those having a genetic predisposition, can be treated with the formulations of the present in a prophylactic attempt to delay or eliminate the onset of the leukemic state. Similarly, those individuals who have already developed cancer and who have been treated to remove the cancer or are otherwise in remission are particularly susceptible to relapse and reoccurrence. As part of a treatment regimen, such individuals can be immunized against the cancer that they have been diagnosed as having had in order to combat a recurrence. Thus, once it is known that an individual has had a type of cancer and is at risk of a relapse, they can be immunized in order to prepare their immune system to combat any future appearance of the cancer.

[0082] Therapeutic approaches based upon the disclosure are premised on a response by a subject's immune system, potentially leading to lysis of leukemia cells. One such approach is the administration of autologous CTLs specific to the complex to a subject with abnormal cells of the phenotype at issue. It is within the skill of the artisan to develop such CTLs in vitro. Generally, a sample of cells taken from a subject, such as blood cells, are contacted with a cell presenting the complex and capable of provoking CTLs to proliferate. The target cell can be a transfectant. These transfectants present the desired complex of their surface and, when combined with a CTL of interest, stimulate its proliferation. Specific production of a CTL is well known to one of ordinary skill in the art. The clonally expanded autologous CTLs can then be administered to the subject.

[0083] In one therapeutic methodology, referred to as adoptive transfer (Greenberg, J. Immunol. 136(5): 1917, 1986; Riddel et al., Science 257: 238, 1992; Lynch et al, Eur. J. Immunol. 21: 1403-1410, 1991; Kast et al., Cell 59: 603-614, 1989), cells presenting the desired complex are combined with CTLs leading to proliferation of the CTLs specific thereto. The proliferated CTLs are then administered to a subject with a cellular abnormality which is characterized by certain of the abnormal cells presenting the particular complex. The CTLs can then lyse the abnormal cells, thereby achieving the desired therapeutic goal.

[0084] The foregoing therapy assumes that at least some of the subject's abnormal cells present the relevant HLA complex. This can be determined very easily, as the art is very familiar with methods for identifying cells which present a particular HLA molecule, as well as how to identify cells expressing DNA of the pertinent sequences, in this case a leukemia associated gene sequence. Once cells presenting the relevant complex are identified via the foregoing screening methodology, they can be combined with a sample from a patient, where the sample contains CTLs. If the complex presenting cells are lysed by the mixed CTL sample, then it can be assumed that a leukemia associated gene is being presented, and the subject is an appropriate candidate for the therapeutic approaches set forth herein.

[0085] Adoptive transfer is not the only form of therapy that is available in accordance with the invention. CTLs can also be provoked in vivo, using a number of approaches. One approach is the use of non-proliferative cells expressing the complex, such as antigen presenting cells. The cells used in this approach can be those that normally express the complex, such as irradiated tumor cells or cells transfected with one or both of the genes necessary for presentation of the complex. Chen et al., Proc. Natl. Acad. Sci. USA 88: 110-114 (1991) exemplifies this approach, showing the use of transfected cells expressing HPV E7 peptides in a therapeutic regime. Various cell types can be used. Similarly, vectors carrying one or both of the genes of interest can be used. Viral or bacterial vectors are especially preferred. The nucleic acid can be incorporated into an expression vector. Expression vectors can be unmodified extrachromosomal nucleic acids, plasmids or viral genomes constructed or modified to enable insertion of exogenous nucleic acids, such as those encoding the present peptides. Nucleic acids encoding these peptides can also be inserted into a retroviral genome, thereby facilitating integration of the nucleic acid into the genome of the target tissue or cell type. In these systems, the gene of interest is carried by a microorganism, e.g., a Vaccinia virus, retrovirus or the bacteria BCG, and the materials defacto “infect” host cells. The cells which result present the complex of interest, and are recognized by autologous CTLs, which then proliferate.

Kits

[0086] The invention also provides isolated proteins and peptides, and antibodies to those proteins and peptides. Kits containing any of the foregoing molecules, alone or in combination, are additionally provided. The foregoing can be used in the diagnosis or treatment of conditions characterized by the expression of the present peptides. The kits can also be used to pulse antigen presenting cells or t-lymphocytes, and, as such, can contain appropriate culture media, culture media supplements such as cytokines, disposable laboratory equipment and the like. Examples of such kit components can be found in the following examples.

[0087] The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the pharmaceutical compositions can be employed in conjunction with other therapeutic compounds.

[0088] The invention in another aspect involves a kit for detecting the presence of the expression of the present polypeptide. Such kits employ two or more of the above-described nucleic acid molecules isolated in separate containers and packaged in a single package. In one such kit, a pair of isolated nucleic acid molecules is provided. In certain embodiments, the pair of isolated nucleic acid molecules are PCR primers.

[0089] The invention also embraces so-called expression kits, which allow the artisan to prepare a desired expression vector or vectors. Such expression kits include at least separate portions of each of the previously discussed coding sequences. Other components can be added, as desired, as long as the previously mentioned sequences, which are required, are Included.

[0090] This invention is further illustrated by the following non-limiting examples.

EXAMPLES Example 1

[0091] The present example illustrates that that the present peptides are capable of inducing a T-lymphocyte response. In this example, antigen presenting cells were pulsed at 150 micrograms of each peptide per 1 million antigen presenting cells with the following amino acid sequences: (1) YLALCLCLI; (SEQ ID NO: 1) (2) AIISGDSPV; and (SEQ ID NO: 2) (3) YIISGDSPV. (SEQ ID NO: 3)

[0092] The antigen presenting cells were then contacted with effector cells at a target:effector ration of 1:60. The effector cells were then contacted with HLA-A2.1 positive ML-2 cells and the cytotoxicity to the ML-2 cells were measured. ML-2 cells are acute myeloid leukemia cells (AML) cells which are HLA-A2.1 positive. The results are set forth in Table 1. TABLE 1 Cytoxicity Cytotoxicity CD33 to ML-1 to AML Antigen Peptide Cells by Patient's Experiment Presenting (SEQ ID Number of Cytokines Bone Number Cells Donor NO:) Stimulations (%) Marrow (%) Control None B, F, G None 0 0, 0, 7 1 DC A 2 2 37 2 DC B 2 2 42 43 3 DC C 2 2 39 63 4 DC D 2 2 29 5 DC E 3 2 37 6 T2 F 1 2 3 21 36 7 T2 G 1 2 3 34 64 8 T2 F 2 2 3 33 56 9 T2 G 2 2 3 35 55

[0093] Table 2 shows the expression levels of different types of effector cells contacted with antigen presenting cells pulsed with examples of peptides of the present invention. Results are shown in mean fluorescence intensity. TABLE 2 CD33 Antigen Peptide Expression levels of: Presenting (SEQ ID Number CD45RO CD95 CD4 CD 8 Cells Donor NO:) of Stims Memory Activation T helper T cytotoxic None F, G None 0  420  24 120 2430 DC C 2 2 1851 110 332 2477 DC D 2 2 2593 172 356 2332 T2 F 2 2 3 2146 1963  77 127 51 49 6267 7793 T2 G 2 2 3 1575 1591 133 152 31 36 8070 8187 T2 F 1 2 1262  99  81 3381 T2 G 1 2 1432 102  57 7796

[0094]FIG. 1A shows IFN-γ release by cytotoxic T-lymphocytes (CTLs) from two different HLA-A2.1+ (shown as diamond or box) donors stimulated by dendritic cells pulsed three times, once a week with the AIISGDSPV (SEQ ID NO:2) peptide. FIG. 1B shows IFN-γ released by CTLs stimulated by T2 cells pulsed as above with the AIISGDSPV (SEQ ID NO:2) peptide. As can be seen from these FIGS., the highest IFN-γ release was observed when the CTLs were stimulated twice. IFN-γ amount is shown in picogram/milliliter. The scale for FIG. 1B is the same as for FIG. 1A.

[0095] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” “more than” and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. In the same manner, all ratios disclosed herein also include all subratios falling within the broader ratio.

[0096] One skilled in the art will also readily recognize that where members are grouped together in a common manner, such as in a Markush group, the present invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group. Accordingly, for all purposes, the present invention encompasses not only the main group, but also the main group absent one or more of the group members. The present invention also envisages the explicit exclusion of one or more of any of the group members in the claimed invention.

[0097] All references disclosed herein are specifically incorporated herein by reference thereto.

[0098] While preferred embodiments have been illustrated and described, it should be understood that changes and modifications can be made therein in accordance with ordinary skill in the art without departing from the invention in its broader aspects as defined in the following claims. 

What is claimed is:
 1. An isolated leukemic antigen comprising a fragment of CD33 antigen (SEQ ID NO:4) or a variant thereof that is capable of stimulating a cytotoxic T-lymphocyte reaction wherein the fragment or variant thereof is 8, 9, 10, 11 or 12 amino acids in length.
 2. The isolated leukemic antigen of claim 1 wherein the fragment of CD33 antigen is immunologically recognized by MHC restricted T-Lymphocytes that are HLA-A2.1 restricted.
 3. The isolated leukemic antigen of claim 2 wherein the fragment of CD33 antigen comprises the amino acid sequence YLALCLCLI (SEQ ID NO: 1), AIISGDSPV (SEQ ID NO: 2) or YIISGDSPV (SEQ ID NO: 3).
 4. The isolated leukemic antigen of claim 1 wherein the variant of the fragment of the CD33 antigen comprises the amino acid sequence YLALCLCLI (SEQ ID NO: 1), AIISGDSPV (SEQ ID NO: 2) or YIISGDSPV (SEQ ID NO: 3) with one or more conservative or nonconservative amino acid substitutions.
 5. The isolated leukemic antigen of claim 1 in combination with one or more co-immunostimulatory molecules.
 6. A method for stimulating an immune effector cell response comprising contacting the isolated leukemic antigen of claim 1 with an immune effector cell thereby stimulating the immune effector cell to respond against the isolated leukemic antigen.
 7. The method of claim 6 wherein the immune effector cell is a naive T-lymphocyte or a memory T-lymphocyte.
 8. The method of claim 6 further comprising contacting the isolated leukemic antigen with an antigen presenting cell wherein the antigen presenting cell contacts the isolated leukemic antigen with the immune effector cell.
 9. The method of claim 8 wherein the antigen presenting cell is a dendritic cell or a T2 cell.
 10. The method of claim 8 wherein the antigen presenting cell contacts the immune effector cell in vitro or in vivo.
 11. The method of claim 10 wherein the isolated leukemic antigen comprises amino acid sequence YLALCLCLI (SEQ ID NO: 1), AIISGDSPV (SEQ ID NO: 2), or YIISGDSPV (SEQ ID NO: 3).
 12. The method of claim 10 wherein the isolated leukemic antigen comprises a variant of the fragment of the CD33 antigen with the amino acid sequence YLALCLCLI (SEQ ID NO: 1), AIISGDSPV (SEQ ID NO: 2) or YIISGDSPV (SEQ ID NO: 3) having one or more conservative or nonconservative amino acid substitutions.
 13. The method of claim 6 wherein contacting the isolated leukemic antigen with the immune effector cell occurs in the presence of an immunostimulatory molecule.
 14. An immune effector cell produced according to the method of claim
 6. 15. The immune effector cell of claim 14 wherein the isolated leukemic antigen comprises the fragment of the CD33 antigen has the amino acid sequence YLALCLCLI (SEQ ID NO: 1), AIISGDSPV (SEQ ID NO: 2), YIISGDSPV (SEQ ID NO: 3) or a variant thereof having one or more conservative or nonconservative amino acid substitutions.
 16. An antigen presenting cell produced by contacting the isolated leukemic antigen of claim 1 with an antigen presenting cell.
 17. The antigen presenting cell of claim 16 wherein the isolated leukemic antigen comprises a fragment of the CD33 antigen with the amino acid sequence YLALCLCLI (SEQ ID NO: 1), AIISGDSPV (SEQ ID NO: 2), YIISGDSPV (SEQ ID NO: 3) or a variant thereof having one or more conservative or nonconservative amino acid substitutions.
 18. An isolated nucleic acid which encodes the isolated leukemic antigen of claim
 1. 19. An isolated nucleic acid which encodes the isolated leukemic antigen of claim
 3. 20. An isolated nucleic acid which encodes the isolated leukemic antigen of claim
 4. 21. A pharmaceutical composition comprising the isolated leukemic antigen of claim
 1. 22. An antibody, or functional fragment thereof, that is capable of binding the isolated leukemic agent of claim
 1. 